The invention relates generally to methods of screening for agents which bind to and/or regulate cellular proteins involved in drug resistance, particularly resistance of tumor cells to chemotherapeutic agents.
Biochemical and genetic approaches are commonly used to identify key aspects of signaling pathways involved in a pathological phenotype. Biochemical approaches characterize intracellular protein components, whereas genetic approaches characterize mutations in cellular components. Pharmacology has long complemented genetic and biochemical approaches by identifying candidate agents that bind to a component of interest. Although candidate agents can be any molecule, they are typically small organic compounds. Small organic compounds often affect polypeptide structure, stability, or function. Additionally, small organic compounds can influence the manner in which proteins interact with each other (Fabbrizio, E., et al., (1999) Oncogene, 18:4357-63). This in trans effector function of small organic molecules offers subtle means by which signaling pathways can be studied or corrected.
Screening molecular libraries of chemical compounds for trans-effector molecules provides another means by which signaling pathways can be characterized. It should be possible to deliver to cells libraries of diverse organic molecules in the form of small peptides. Peptides have been used in in vitro library screens against known target proteins (Wong, D. W. and Robertson, G. H., (1998) Ann. N. Y. Acad. Sci., 864, 555-557; Bremnes, T., et al.,.(1998) Immunotechnology, 4, 21-8). Peptide libraries expressed in phage have been used to isolate high affinity aptamers that bind to known target molecules. Such binding can be shown to influence protein function in vitro. Chemically synthesized libraries of peptides also have been made and screened in a variety of in vitro assays with similar levels of efficacy. However, it has not been possible, to screen peptides against entire pathways within living cells.
An approach based on screening peptides against entire pathways requires knowledge of the stimulus that activates the disease and a phenotypic outcome. For instance, the stimulus can be environmentally determined, such as the response to a cytokine, or can be constitutively present, such as a mutated protein that continuously signals a mitogenic response leading to an oncogenic phenotype. The phenotypic outcome can be considered movement towards, or away from, the disease state using as a genetic selection tool any of a number of surrogate indicators of phenotype. The closest attempts at such are screens of cDNA libraries for complementation in mammalian cells.
It would be particularly valuable to screen for peptides against pathways which lead to the loss of sensitivity to chemotherapeutic drugs in tumor cells. The loss of sensitivity to chemotherapeutic drugs results in resistance to multiple chemotherapeutic drugs. This resistance is termed multi-drug resistance (MDR) and arises with a variety of therapeutic agents in a wide range of malignancies. Multiple drug resistance, and hence tumor cell selection, outgrowth, and relapse, leads to morbidity and mortality in cancer patients.
One mechanism used by tumor cells to acquire MDR is up-regulation of P-glycoprotein (P-gp), the product of the MDR-1 gene. Such upregulation is associated with the MDR phenotype of many human cancers, including a wide variety of solid tumors (Kaye, S. B., (1998) Curr. Opin. Oncol., 10 Suppl 1, S15-19) and certain leukemias and lymphomas (Hart, S. M. et al, (1993) Leuk Lymphoma, 11, 239-248; Yamaguchi, M. et al., (1995) Cancer, 76, 2351-2356). P-gp is a transmembrane protein that functions as an energy-dependent efflux pump whose normal function is to transport metabolites and to provide protection against cytotoxic substances (Bello-Reuss, E. and Ernest, S., (1994) Am. J. Physiol., 267, C1351-1358; Chin, K. V., et al., (1990) Cell Growth Differ., 8, 361-365). Over-expression of P-gp results in the expulsion of intracellular anticancer drugs and the consequent establishment of resistance to those compounds. Such drug resistance has been demonstrated in tumor cells upon exposure to cytotoxic drugs such as Taxol (Parekh, H., Wiesen, K. and Simpkins, H. (1997) Biochem. Pharmacol., 53, 461-470), as well as with many other chemotherapeutic agents (Fardel, O., et al., (1994) Eur. J. Biochem., 219, 521-528). Other proteins implicated in MDR are regulators of apoptosis (i.e., Bcl-2 and Bcl-xL) and LRP (lung resistance protein). The LRP protein has been implicated in multidrug resistance in ovarian carcinoma, metastatic malignant melanoma, and acute myeloid leukemia (Leith, C., (1998) Curr. Opin. Hematol., 5, 287-291).
Accordingly, the cellular components involved in rendering cells resistant to chemotherapy is of paramount interest, and it is an object of the invention to provide proteins and related molecules involved in MDR. It is a further object of the invention to provide recombinant nucleic acids involved in MDR, and expression vectors and host cells containing the nucleic acid encoding proteins involved in MDR. A further object of the invention is to provide methods for screening for antagonists and agonists of proteins involved in MD.
In accordance with the objects outlined above, the present invention provides methods and compositions for screening for agents that confer or ameliorate MDR. Accordingly, the invention provides bioactive agent, such as resistance conferring peptides that confer a MDR phenotype and drug candidates that ameliorate the MDR phenotype.
In one aspect, the invention provides methods for screening for bioactive agents that confer multi-drug resistance on a cell. The methods comprise the steps of a) introducing a retroviral library of randomized candidate peptide into a plurality of cells, wherein each of said peptides comprises a different peptide sequence; b) screening the plurality of cells for a 6 cell exhibiting a MDR phenotype, wherein the MDR phenotype is due to the presence of a bioactive agent. The methods may also include the steps of c) isolating the cell(s) exhibiting the MDR phenotype; and, d) isolating a resistance conferring peptide from the cell(s).
The invention further provides methods for isolating a protein of a resistance pathway.
In a further aspect, the invention provides methods for screening for a bioactive agent capable of binding a protein of a multi-drug resistance pathway. The methods comprise the steps of a) combining a protein of a multi-drug resistance pathway and a candidate bioactive agent; and b) determining the binding of the candidate agent to the protein.
The invention further provides methods of identifying a drug candidate comprising the steps of a) contacting a protein of a multi-drug resistance pathway and a resistance conferring peptide; and b) detecting a decrease in the binding of the peptide to the protein in the presence of the candidate drug, thereby identifying a drug candidate.
In a further aspect, the invention provides methods for identifying a bioactive agent capable of modulating, the activity of a protein of a multi-drug resistance pathway. The methods comprise the steps of a) adding a candidate bioactive agent to a plurality of cells, wherein each of said cells expresses a protein of a multi-drug resistance pathway; b) adding a chemotherapeutic agent to said plurality; and c) determining the effect of the candidate bioactive agent on resistance to the chemotherapeutic agent.
In an additional aspect, the present invention provides methods of treating a multidrug resistance associated disease comprising administering compositions of modulators of proteins of multi-drug resistance pathway.